Bispecific antibody and antibody conjugate for tumour therapy and use thereof
a tumour and antibody technology, applied in the field of specific antibodies, can solve the problems of increased half-life, reduced toxicity, and reduced manufacturing efficiency
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example 2
ansformation of BL21 (DE3)
[0111]The plasmid was transformed into Escherichia coli DH5a competent cell line, positive clones were selected, cultured in LB medium (3 mL) containing 100 μg / ml ampicillin at 37° C. overnight, the culture solution was centrifuged at 5000 rpm and the supernatant was discarded. The plasmids were extracted from E. coli by using plasmid extraction kit from Qiagen and the expression vector was obtained.
example 3
n and Purification of Anti-MUC1-Anti-CD16
[0112]The purified pETDuet-bsAb expression vector was transformed into E. coli BL21 (DE3) strain, and positive clones were selected and cultured in LB medium (3 mL) containing 100 μg / mL ampicillin overnight at 37° C., and then transferred to 300 mL LB containing 100 μg / mL ampicillin, and the cells were cultured at 37° C. until the OD600 was at 0.6-0.8. IPTG was added at a final concentration of 0.05 mM, and the culture was induced at 16° C. for 16 hours. The culture was centrifuged at 4000 rpm, and the supernatant was discarded. The precipitate was added to a solution of 20 mM Tris-HCl, pH 8.0, 25% sucrose, 1 mM EDTA in a weight-to-volume ratio of 1:4, resuspended, ice-bathed for 30 min, and centrifuged at 4° C., 8500 g, for 20 minutes. The supernatant was taken. The precipitate was added at a weight ratio of 1:5 to solution containing 5 mM MgCl2 and 1 mg / mL lysozyme, ice-cooled for 20 min, and centrifuged at 4° C., 8500 g, for 20 minutes. Th...
example 4
c Antibody In Vitro Binding to MUC1 Test M
Method:
1). Flow Cytometry Detects the Binding of Bispecific Antibody to MUC1
[0119]1. MUC1 positive cells LS174T, HT29, SKOV3 and negative cells CHO, HepG2 cells were cultured in vitro; 0.25% trypsin was digested into single cells, and after centrifugation at 1000 rpm for 10 min, the cell pellet was collected and resuspended in ice-cold PBS+0.2% BSA.
[0120]2. Centrifuged at 4° C., 1000 rpm, for 5 minutes, discarded the supernatant
[0121]3. Resuspend in ice-cold PBS+0.2% BSA and configured to a cell suspension with a concentration of 2*106 / mL.
[0122]4. The primary antibodies (mouse anti-MUC1 / CD66e mAb (1:200); bsAb (10 ug / mL)) were added separately as per the following table and then were incubated for 1 hour at 4° C.
Tube numberPrimary antibodiesSecondary antibodiesANoneGoat-Anti-mouse IgG-FITC (1:500)BAnti-MUC1 (1:200)Goat-Anti-mouse IgG-FITC (1:500)CNoneAnti-His-FITC (1:500)DbsAb (10 ug / mL)Anti-His-FITC (1:500)
[0123]5. Added 5 mL ice-cold PBS+0...
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